Ketone bodies predominantly consist of the D-enantiomer of β-hydroxybutyrate (D-βHB), which is the major circulatory KB, and acetoacetate (Robinson & Williamson, 1980). KBs are synthesized primarily from the hepatic conversion of fatty acids during physiological states of low carbohydrate availability, such as starvation, ingestion of a very low carbohydrate, high-fat diet, and prolonged exercise to provide an alternative fuel source for extrahepatic tissues (e.g., skeletal muscle, brain, and heart). Postprandial blood KB concentration is ∼0.1–0.2 mmol/L, whereas hyperketonemia is defined as concentrations above 0.5 mmol/L (Robinson & Williamson, 1980). The oral ingestion of ketone supplements, specifically salts and esters, have shown to induce hyperketonemia within 30 min (Stubbs et al., 2017), thus allowing the effect of KBs to be delineated from the interference to changes in substrate availability and metabolism resulting from alternative ketogenic strategies.

The impact of acute hyperketonemia on endurance performance remains equivocal, with beneficial (Cox et al., 2016), detrimental (Leckey et al., 2017; O’Malley et al., 2017), and trivial (Evans & Egan, 2018; Rodger et al., 2017) effects reported. This is likely due to differences in methodology, particularly the use of varying forms of ketone supplements and the manipulation of carbohydrate availability before and during the performance test. In addition, the previously mentioned studies were unlikely to be of sufficient duration and intensity to deplete skeletal muscle glycogen stores, which may be required to elucidate an ergogenic effect of acute hyperketonemia. For example, the longest trial consisted of a 4-min cycling time trial (TT) preceded by 90 min of cycling at 80% of the power-eliciting secondary ventilatory threshold (VT2; Rodger et al., 2017). Therefore, further investigations are warranted to elucidate the effect of different ketone supplements without the interference of concomitant carbohydrate supplementation on endurance performance during prolonged, high-intensity protocols.

To explore the effect of acute hyperketonemia on a ∼30-min cycling TT performed following a glycogen-depletion phase, we used the racemic βHB precursor, R,S-1,3-butanediol (BD). R,S-1,3-butanediol is a widely available nontoxic dialcohol and component part of the R,S-1,3-butanediol acetoacetate diester (Leckey et al., 2017). Following ingestion, BD is passively absorbed in the gut (Shivva et al., 2016) and subsequently increases blood R- and S-BD, before rapidly undergoing hepatic conversion to the isotopic enantiomers, D-βHB and L-βHB (Desrochers et al., 1992). The ingestion of BD has shown to increase blood D-βHB concentrations to ∼1.0 mmol/L within 30 min in rats (Kesl et al., 2016); however, there is currently no published literature on the dose response of BD in humans or its effect on endurance performance.

Preliminary Testing and Familiarization

Participants presented to the laboratory on two occasions prior to the experimental trials. On the first visit, participants arrived at a time of convenience, having fasted for a minimum of 4 hr and refraining from caffeine, alcohol, and strenuous exercise for the preceding 24 hr. Participants’ body mass (shorts only) and height were measured. To determine VO2peak, VT2, and maximal power output (Wmax), participants performed a continuous incremental cycling protocol on an electromagnetically braked cycle ergometer (Lode Excalibur Sport, Groningen, the Netherlands) commencing cycling at 95 W with increments of 35 W every 3 min until either (a) volitional exhaustion or (b) cadence could not be maintained above 60 rpm. Ratings of perceived exertion (RPE; Borg 6–20 scale; Borg, 1982) and heart rate (HR) using short-range telemetry (Garmin Fenix 3; Garmin Ltd., Olathe, KS) were noted during the final 30 s of each stage. The expired gas was collected and analyzed continuously using a computerized metabolic system with mixing chamber (TrueOne 2400; Parvo Medics, Salt Lake City, UT), and Wmax was calculated according to the formula: Wmax = Wfinal + (t/T) × Winc, where Wfinal is the power output (W) of the final completed stage, t is the time achieved in the final uncompleted stage (s), T is the duration of each stage (180 s), and Winc is the workload increment (35 W). The power-eliciting VT2 was determined using the V-slope method (Beaver et al., 1986) by two researchers in a pro-rata manner and VO2peak was determined by the highest averaged 30 s. To familiarize participants with the TT, participants remounted the cycle ergometer after 15–20 min of rest, which was switched from hyperbolic to linear mode and commenced a TT equivalent to 7 kJ/kg (∼25–35 min). The power output in linear mode is cadence (rpm) dependent, with power (W) calculated according to the formula: W = L × (rpm)2. The linear factor (L) was calculated to elicit a power output of 70% Wmax at an rpm of 90. For all cycling tests, bike dimensions were set to the participants’ preferences and were repeated for subsequent trials. The participants returned after 3–7 days to perform a familiarization protocol, which involved completing the requirements of the experimental trial without the ingestion of BD, fluid restriction, or blood collection (described below).

Pilot Test

To investigate the dose response of BD on blood D-βHB concentration and tolerability, a pilot test was conducted prior to the experimental trials. Six healthy, recreationally active males undertook four different dosing protocols, which included the provision of BD within two 200-ml boluses of an artificially sweetened, flavored drink separated by 1.5 hr. These doses were a placebo of 0 + 0 g/kg (PLA), medium dose of 0.5 + 0 g/kg (M-BD), high dose of 0.7 + 0 g/kg (H-BD), and a split dose of 0.35 + 0.35 g/kg (Spl-BD) of BD. All tests commenced between 06:00 and 08:00 hr with participants in a fasted state, and D-βHB was measured across 3.5 hr following the ingestion of the initial bolus to align with time points of the experimental trial (Figure 1). We concluded that BD split into two 0.35 g/kg boluses elicited maximal D-βHB concentration and minimal side effects compared with BD given as a single, larger bolus of 0.5 or 0.7 g/kg, which tended to result in nausea, euphoria, and dizziness.

Experimental Trial

Using a repeated-measures, randomized, crossover design, participants were randomly (www.randomizer.org) assigned to either PLA or BD (proportional distribution of optical isomers unknown; product code 02-59620; Penta Manufacturing Ltd., Livingston, MT). Participants were not informed of their trial allocation. However, due to the difficulty masking the bitter taste of BD, achieving successful blinding was deemed unlikely. For the day prior to each experimental trial, participants were prescribed a diet consisting of 6 g/kg of carbohydrate based on their dietary preferences by an experienced registered dietitian, which was to be consumed by 23:00 hr, and were asked to avoid caffeine and alcohol for the day prior to each experimental trial. Compliance was confirmed using an image-assisted weighed dietary record reported remotely in real time through a mobile phone application (WhatsApp, Mountain View, CA; Facebook, Menlo Park, CA). In addition, participants were asked to refrain from strenuous exercise for the preceding 48 hr and to consume 500 ml of water prior to arrival. The purpose of standardizing carbohydrate intake and exercise before each trial was to normalize carbohydrate availability to promote similar rates of muscle glycogen utilization prior to the TT. This was estimated at ∼50% based on our participant characteristics and exercise protocol (Areta & Hopkins, 2018). Participants arrived at the laboratory between 07:00 and 08:00 hr having fasted from 23:00 hr the previous day. An indwelling intravenous teflon catheter (18G; Terumo Corp., Tokyo, Japan) was inserted into the antecubital vein for serial blood sampling. This was followed by the ingestion of a bolus of 0 or 0.35 g/kg BD within a 2 ml/kg artificially sweetened, orange-flavored drink (Thriftee; Hansells Food Group Ltd., Auckland, New Zealand), that is, presupplement. After 30 min (i.e., pre-exercise), participants commenced steady-state (SS) cycling at the power output eliciting 85% of their VT2 (240.8 ± 28.3 W; 62.0% ± 2.4% Wmax; 73.0% ± 5.2% VO2peak) for 85 min. Every 20 min, HR and expired gas were collected, with participants providing their RPE. After 60 min of cycling (i.e., 60-min exercise), participants ingested a second bolus of PLA or 0.35 g/kg BD according to their trial allocation. Following completion (i.e., post-SS), participants rested for 5 min, then were instructed to complete the TT (as previously mentioned) as fast as possible while remaining in a seated position. Participants were blinded to their power output and elapsed time; however, they were notified at each quarter of completion and were counted down from 100 kJ in 10 kJ decrements. No respiratory or blood samples were collected during the TT; however, HR was collected at each quarter of completion. All trials were conducted by the same researcher and standardized encouragement was provided. Fluid intake was restricted to 2 ml/kg of water every 15 min during the SS and TT phases. Following the TT (i.e., post-TT), participants removed wet clothing and towel dried themselves prior to having their body mass measured. Participants then completed a customized questionnaire adapted from published sources (Clarke et al., 2012; Pfeiffer et al., 2009), including 27 items pertaining to systemic (13), upper abdominal (six), and lower abdominal (eight) symptoms, and prior tested by two experienced registered dietitians for understanding and literacy. Participants were prompted for additional symptoms not stated on the questionnaire and were asked to identify their trial allocation. They were then provided with 5 ml/kg of water and rested for 60 min prior to departing. All exercise trials throughout the study were performed in standard laboratory conditions of 18.3 ± 0.6°C and 67.6% ± 7.4% relative humidity and were separated by 7–10 days.

Data Analysis

All data are expressed as mean ± SD unless otherwise stated. Data were checked for normality as indicated by the Shapiro–Wilk score, and where appropriate, statistical analysis was performed on the logarithmic transformation of the data. Paired t tests were used to compare TT duration, average TT power output and HR, and change in body mass between trials. A two-way (Trial × Time) repeated-measures analysis of variance was performed for glucose, D-βHB, lactate, cardiorespiratory, and RPE data (IBM SPSS Statistics software, version 21; IBM Corp., Chicago, IL). If Mauchly’s test of sphericity was violated, adjustments to the degrees of freedom were made for the analysis of variance using Greenhouse–Geisser ϵ. Where a significant effect was observed, post hoc analysis was conducted using Student’s paired t tests with Holm–Bonferroni adjustments for multiple comparisons applied to the unadjusted p value to locate specific differences. Significance level was accepted at an alpha of p d effect sizes (±90% confidence limits) were estimated using a purpose-built spreadsheet (Hopkins, 2006), with effect size thresholds set at <0.2, >0.2, >0.6, >1.2, >2.0, and >4.0 for trivial, small, moderate, large, very large, and extremely large effects, respectively (Hopkins et al., 2009). However, as Cohen’s d may overestimate the effect of BD on D-βHB concentration compared with ketone supplements with higher bioavailability, we used a novel approach to determine the magnitude of effect whereby the possible range of change was transformed into a full scale of deflection (FSD; Hopkins, 2010). The deflection (±90% confidence limits) was estimated by the difference in D-βHB concentration between the PLA and BD trials for each time point and the range was calculated by subtracting D-βHB concentration of the PLA trial for each time point from 3.5 mmol/L, which is approximately the highest D-βHB concentration reported during exercise of a similar intensity following the ingestion of a ketone supplement (Cox et al., 2016). Each range was set at 0–100%, and the magnitude thresholds were defined as >10%, >30%, >50%, >70%, and >90% for small, moderate, large, very large, and extremely large effects, respectively. If the 90% confidence limits overlapped 0 for either effect size statistics, the magnitude of effect was deemed unclear.

Tolerability

There was no difference in the change of body mass (corrected for fluid intake) between trials (PLA, −2.14 ± 0.48 kg; BD, −2.07 ±0.42 kg; p = .15, d = 0.13 [−0.02, 0.28]). Within the BD trial, two participants experienced transient symptoms of low levels of nausea, euphoria, and dizziness, which they related to a state of alcohol intoxication. Five participants reported low to moderate levels of belching and burping, and one participant reported severe abdominal pain. No participants reported similar symptoms during the PLA trial. Everyone disliked the taste of BD, which resulted in retching in four participants. Everyone correctly identified their trial allocation, which was likely due to the difficulty masking the taste of BD and subsequent gastrointestinal effects.

Discussion

To our knowledge, this is the first study investigating the effects of BD ingestion on performance. Despite BD increasing blood D-βHB concentration, no other significant differences in metabolic, cardiorespiratory, or performance variables were observed. These findings support previous work suggesting ketone supplements eliciting blood D-βHB concentrations up to ∼1 mmol/L do not benefit endurance performance. Furthermore, we found BD to elicit gastrointestinal distress, in particular belching and burping, as well as symptoms of nausea, euphoria, and dizziness in some participants.

In this study, BD paralleled the bioavailability of other racemic ketone supplements. The use of 2 × 0.35 g/kg of BD increased blood D-βHB concentration to levels during exercise (0.44–0.79 mmol/L) similar to the ingestion of 2 × ∼0.4 g/kg of a racemic βHB salt solution (total of 24–37 g D,L-βHB; Evans et al., 2018; Rodger et al., 2017) and 2 × 0.25 g/kg of a R,S-1,3-butanediol acetoacetate diester (Leckey et al., 2017). It is likely these racemic ketone supplements also increased blood L-βHB; however, as L-βHB largely resides intracellularly in very low concentrations and does not directly contribute to energy production, it is not considered an important substrate in this context (Desrochers et al., 1992; Stubbs et al., 2017). Notably, our peak D-βHB concentrations occurred at 1-hr post-TT, which was substantially higher than values observed in our pilot test; however, post-exercise ketogenesis was a likely contributor (Koeslag et al., 1980). Nevertheless, our blood D-βHB concentrations were markedly lower compared with the ingestion of the nonracemic D-βHB R-1,3-butanediol monoester, which can increase D-βHB to ∼2.5–3.5 mmol/L during exercise (Cox et al., 2016). This highlights the low bioavailability and differences in componentry of BD and other racemic ketone supplements compared with the D-βHB R-1,3-butanediol monoester.

Following the onset of exercise, blood D-βHB concentration declined and plateaued until after the second bolus of BD ingestion at 60-min exercise. Compared with our pilot data in a separate resting population, blood D-βHB concentration was ∼0.2–0.3 mmol/L lower from 30-min exercise to post-TT. Collectively, this suggests D-βHB was continuously released into the circulation, while being taken up by muscle cells through monocarboxylate transporters (Halestrap & Wilson, 2012). Although there was not a shift in the RER toward βHB and acetoacetate’s respiratory quotient of 0.89 and 1.0, respectively, this effect appears to be abrogated at exercise intensities above 60% VO2max (Evans et al., 2018). An earlier study estimated D-βHB oxidation rates by comparing area under the curve for blood D-βHB concentrations between resting and exercising conditions following the ingestion of D-βHB R-1,3-butanediol monoester (Cox et al., 2016); however, as we did not measure resting D-βHB concentrations for our cyclists, we could not compare D-βHB oxidation rates. Importantly, the validity of these calculations have not been confirmed using direct calorimetry and may be compromised due to the inclusion of unclear D-βHB volume distribution values, which could also change following exercise or ketone supplement ingestion (Frayn, 1983).

Clearly, the physiological and nutritional conditions suitable for ketone supplements to augment performance remain difficult to identify. Considering the potential for hyperketonemia to downregulate glycolysis and suppress adipocyte lipolysis, the optimal range of KB concentration to enhance substrate provision and energy production remains obscure. This is exacerbated by differences in the bioavailability of various ketone supplements and measurement discrepancy of point-of-care versus laboratory-based methods (Guimont et al., 2015; Leckey et al., 2017), thus making comparisons between studies difficult. For example, following the ingestion of the D-βHB R-1,3-butanediol monoester within a carbohydrate drink, plasma D-βHB was maintained above ∼2.0–3.0 mmol/L and improved performance during a preloaded 30-min cycling TT by ∼2% compared with the ingestion of an isoenergetic carbohydrate-only drink (Cox et al., 2016). However, these performance benefits have not been replicated and may be abrogated in exercise trials 90 min or less proceeding the ingestion of recommended carbohydrate intakes (Cox et al., 2016; Evans & Egan, 2018; Leckey et al., 2017). It is possible that the interaction between KB concentration and carbohydrate availability has a mediating role on KB metabolism (Chari & Wertheimer, 1954), with high rates of KB oxidation requiring high-carbohydrate availability to maintain the anaplerotic flux in the tricarboxylic acid cycle (Russell & Taegtmeyer, 1991a, 1991b). However, how this translates to real-world performance is yet to be elucidated.

The ingestion of BD also led to symptoms synonymous with low levels of alcohol intoxication within two participants. Typically, BD is rapidly converted to βHB by hepatic alcohol and aldehyde dehydrogenases (Desrochers et al., 1992). However, these steps are rate limiting and may be influenced by previous ethanol exposure (Münst et al., 1981). In this study, participants habitually consumed low levels of alcohol (∼1.5 standard drinks per week); therefore, their maximal capacity to metabolize BD into βHB could have been below the dose ingested, resulting in the accumulation of BD in the circulation. Furthermore, five participants reported moderate to severe gastrointestinal symptoms and despite it not effecting TT performance, it is a deterrent to the use of some ketone supplements (Evans et al., 2018; Leckey et al., 2017). Arguably, these effects may have been exacerbated by ingesting BD in a fasted state, which is not reflective of real-world conditions. We also did not observe significant elevations in RPE as in the case of R,S-1,3-butanediol acetoacetate diester, which were likely due to the resultant gastrointestinal distress (Leckey et al., 2017). However, there was a trend toward a moderate increase in RPE nearing the end of SS cycling in the BD trial, which may have been due to approaching an upper limit of BD ingestion. Collectively, this would limit the application of some ketone supplements to endurance events where exogenous substrate provision and management of gastrointestinal distress are critical factors for performance.

Conclusions

Similar to other ketone supplements eliciting blood D-βHB concentrations up to ∼1 mmol/L, BD does not benefit endurance performance. However, it is uncertain whether this absence of effect persists in events for >2–3 hr. BD may also induce symptoms related to a low level of alcohol intoxication, including nausea, euphoria, and dizziness, as well as moderate to severe gastrointestinal symptoms, suggesting that ingestion should be avoided in higher doses. Further attempts to identify the ergogenic properties of ketone supplements need to focus on products with higher bioavailability and their interaction with varying levels of carbohydrate availability.

Acknowledgments

The study was designed by D. M. Shaw, F. Merien, A. Braakhuis, D. Plews, P. Laursen, and D. K. Dulson; data were collected by D. M. Shaw; data interpretation and manuscript preparation were undertaken by D. M. Shaw, F. Merien, A. Braakhuis, D. Plews, P. Laursen, and D. K. Dulson. All authors approved the final version of the article. The authors would like to thank the participants for their effort, cooperation, and humor. The authors do not have any conflicts of interest.

If the inline PDF is not rendering correctly, you can download the PDF file here.

*Shaw, Plews, Laursen, and Dulson are with the Sports Performance Research Institute New Zealand (SPRINZ), Auckland University of Technology, Auckland, New Zealand. Merien is with AUT Roche Diagnostics Laboratory, School of Science, Auckland University of Technology, Auckland, New Zealand. Braakhuis is with the Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand.